PHOSPHORYLATION AND ACTIVATION OF THE DNA-BINDING ACTIVITY OF PURIFIED STAT1 BY THE JANUS PROTEIN-TYROSINE KINASES AND THE EPIDERMAL GROWTH-FACTOR RECEPTOR

The activation of Janus protein-tyrosine kinases (Jaks) and the subseq uent phosphorylation and activation of latent signal transducers and a ctivators of transcription (Stats) are common elements in signal trans duction through the cytokine receptor superfamily. To assess the role and specificity of Jaks in Stat activation, we have utilized baculovir us expression systems to produce Stat1 and the Jaks. Co-expression of Stat1 with Tyk2, Jak1, or Jak2 resulted in the specific tyrosine phosp horylation of Stat1 at Tyr(701), the residue phosphorylated in mammali an cells stimulated with interferon gamma. Alternatively, Stat1, purif ied to apparent homogeneity from insect cell extracts, was phosphoryla ted at Tyr(701) Jak immune complex kinase reactions. Phosphorylation o f purified Stat1 was necessary and sufficient for the acquisition of D NA binding activity. The specificity in both systems was indicated by the inability of a Jak2 catalytically inactive mutant (Jak2-Glu(882)) or the Tec protein-tyrosine kinase to phosphorylate Stat1. However, im mune complex purified epidermal growth factor receptor was capable of phosphorylating purified Stat1 at Tyr(701) and activating its DNA bind ing activity in in vitro reactions.